The amino-acid sequence of pig-heart cytoplasmic malate dehydrogenase (sMDH) will be compared to that of lactate dehydrogenase (LDH) at both the primary and three-dimensional levels. Various chemical probes will be used to ascertain features of the solution topography of the sMDH molecule. The amino-acid sequence of pig-heart mitochondrial malate dehydrogenase (mMDH) will be completed and compared with that of sMDH and LDH. Similar conformational analyses will be carried out on this enzyme as well. The sequence of chicken-heart mMDH will be determined comparatively as this enzyme appears to have superior crystallographic properties as compared to the pig-heart one. The amino-acid sequence of beta-hydroxyacyl CoA dehydrogenase will be determined by isolation and characterization of the fragments produced by CNBr, trypsin and chymotrypsin. These data will be used to interpret the X-ray studies now in progress and to deduce the extent of similarity between this enzyme and mMDH. D-3-Phosphoglycerate dehydrogenase (PGD) will be isolated from pig liver by both non-specific and affinity-chromatography techniques. Blue dextran attached to Sepharose seems the most promising route as it has been used successfully to isolate several of the NAD ion-dependent hydrogenases from heart tissue. Characterization will include determination of molecular weight, subunit structure, amino-acid composition, isoelectric point and kinetic properties. Sequence analysis will be carried out by techniques similar to that employed for beta-HADH. The objective in examining this member of the NAD ion-dependent dehydrogenase family is to elucidate how the regulatory properties of a branch-point enzyme have been incorporated into a pre-existing structure. The entity responsible for the transport of L-malate across the mitochondrial membrane will be isolated by affinity chromatography in dilute detergents. It will be characterized molecularly and used to prepare liposomes that can be used to study the $ interrelation of s- and m-MDH via this malate transport system. Such a reconstituted system can provide information about how members of a metabolic unit, in this case the malate shuttle pathway, are regulated physiologically.